Description of the Prior Art
Measurement of the severity of physiological response to lead exposure is important for the public health goals of treatment and prevention of lead poisoning. Lead has long been known as a potent neurotoxin which can cause developmental disabilities, behavioral disturbances and mental retardation in children. In adults, the symptoms of plumbism often resemble schizophrenia. Lead poisoning in children and adults is curable, although the neurological damage in children is irreversible.
Sources of lead include lead-based paint or lead solder in pipes, airborne lead from automobile and industrial emissions and lead from occupational sources. Lead can be ingested through the lungs or digestive tract and once ingested, lead accumulates in bones and teeth. Long-term chelation therapy can be used to remove lead from bone tissue. However, if lead poisoning is untreated, the sequestered lead in bone tissue can be reintroduced into the blood stream during periods of bone resorption such as osteoporosis, pregnancy and lactation. In the latter two periods, lead is presented as an irreversible neurotoxin to the fetus and infant. For those reasons, early detection and prevention of lead exposure is essential, especially for small children.
The Centers for Disease Control (CDC) in Atlanta, Ga. have established a guideline which consists of a threshold level of blood lead at which medical intervention is required. The CDC recently lowered the guideline from .gtoreq.25 ug/dL to .gtoreq.10 ug/dL in response to scientific evidence that neurotoxicity in children and infants can occur at very low levels of lead exposure. The CDC has also recommended routine screening of all children for exposure to lead (Preventing lead poisoning in young children: A statement by the Centers for Disease Control--October 1991. Atlanta, Ga.: Centers for Disease Control; 1991.; S. K. Cummins and L. R. Goldman, Pediatrics 90:995-997 (1992)). With the exception of atomic absorption spectroscopy, current diagnostic methods for measuring blood lead are not sufficiently sensitive, reliable or accurate to routinely measure such low levels of blood lead. In addition, the present methods, including atomic absorption spectroscopy, suffer from limitations of expense and time-consuming procedures.
Atomic absorption spectrometry, for example, is a sensitive and accurate method for quantitating blood lead concentration. This method can be used to measure blood lead concentrations below 10 ug/dL; however, atomic absorption spectrometry requires expensive instrumentation and high levels of technical expertise, and therefore cannot be performed in the field (B. E. Jacobson, G. Lockitch, and G. Quigley, Clin. Chem. 37:815-519 (1991)). Additional disadvantages of this method include high expense per test and a time delay between sampling and acquisition of test results. Due to the expense involved and instrumentation required, atomic absorption spectroscopy is not optimal for use as a routine screening assay for field use.
Another method for measuring blood lead concentration is the Erythrocyte Protoporphyrin (EP) test (also known as the Zinc Protoporphyrin test) which is based on the proportional increase in concentration of erythrocyte protoporphyrin in response to lead exposure. Erythrocyte protoporphyrin is a precursor of heme that accumulates as a result of high lead levels interfering with normal heme synthesis. At blood lead concentrations .gtoreq.40 ug/dL, the concentration of erythrocyte protoporphyrin increases exponentially, therefore an erythrocyte protoporphyrin concentration above a specified threshold provides an indication of excessive lead exposure (M. R. DeBaun and H. C. Sox, Jr., Pediatrics, 88:121-131 (1991)). Low sensitivity is the main disadvantage of the EP method; the test is not sensitive to blood lead concentrations below 25 ug/dL (M. D. McElvaine, H. G. Orgach, S. Binder, L. A. Blanksma, E. F. Maes and R. M. Krieg, J. Pediatrics 119:548-550 (1991)). Although inexpensive and easy to perform, the EP test is not a good screening assay because its sensitivity does not allow measurement of the low blood lead levels that the CDC has targeted.
The European Standardized Method (ESM) of determining blood lead concentrations is based on lead inhibition of the enzyme activity of porphobilinogen synthase (PBGS) (also known as delta-aminolevulinic acid dehydratose) (A. Berlin and K. M. Schaller, J. Clin. Chem. Clin. Biochem. 12:389-380 (1977)). PBGS catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA) to form porphobilinogen (PBG) and is extremely sensitive to the presence of divalent lead. The inhibitory effect of lead on PBGS is well established and PBGS has been identified as the most sensitive early indicator of lead poisoning in humans (E. K. Jaffe, S. Bagla and P. A. Michini, Biol. Trace Elem. Res. 28:223-231 (1991)). Inhibition of PBGS by lead causes accumulation of ALA which is believed to be responsible for the neurologic damage in individuals exposed to lead and contributes to the anemia associated with lead poisoning (R. Taylor, J. NIH Res. 2:57-60 (1990); K. Tomokuni, M. Ichiba and Y. Hirai, Toxicol. Lett. 59:169-173 (1991); H. P. Monteiro, D. S. P. Abdalla, O. Augusto and E. J. H. Bechara, Arch. Biochem. Biophys. 271:206-216 (1989)).
The European Standard Method is based on measurement and comparison of PBGS enzyme activity as inhibited by lead and PBGS enzyme activity as reactivated by heat and dithiothreitol (T. Sakai, S. Yanagihara and K. Ushio, Clin. Chem. 26:625-628 (1980)). The ratio of inhibited PBGS activity to reactivated PBGS activity is directly proportional to the blood lead concentration (J. L. Granick, S. Sassa, S. Granick. R. D. Levere and A. Kappas, Biochem. Med. 8:149-159 (1973); R. A. Mitchell, J. E. Drake, L. A. Wittlin and T. A. Rejent, Clin. Chem. 23:105-111 (1977)). The analysis of PBGS activity is based on colorimetric determination of the enzyme product, PBG, through reaction with Ehrlich's reagent. ESM is sensitive to low blood lead concentrations; however, the main disadvantage of the ESM is its irreproducibility caused by factors such as pH, temperature, buffer, variable metal contamination and concentrations of other blood proteins (E. K. Jaffe, S. Bagla and P. A. Michini, Biol. Trace Elem. Res. 28:223-231 (1991) and references therein). In addition, the ESM assay time is long (approximately ninety minutes) and because the assay requires chemical laboratory equipment, the test cannot be easily performed in the field. Although sensitive and inexpensive, the ESM assay is not a good screening method for low blood lead concentrations because it is not considered a reliable method for assessing lead exposure.
The assay described in the International Patent application PCT/US92/05658, entitled "Lead Assays", involves use of PBGS as a biodetector molecule to measure lead concentrations in biological as well as nonbiological materials. For this assay, PBGS can be isolated from tissue or purchased commercially. In carrying out this assay, nitric acid is used to extract elemental lead from any sample. The lead concentration of the sample is then determined either by measuring the PBGS activity after exposure to the sample or by measuring the amount of PBGS complexed to lead after exposure to the sample.
In the assay described in PCT/US92/05658, PBGS activity is measured by monitoring the product PBG formed or the substrate ALA consumed using either chemical labelling of or antibody binding to the product or substrate. Alternatively, the amount of PBGS bound to lead is measured using antibodies that bind specifically to different forms of the PBGS-lead complex. A purported advantage of using PBGS as a biological detector is the ability to test any solution for lead content. However, an apparent disadvantage of this method as a screening test for human blood is the lack of specificity to the human subject since PBGS from any species can be used.
The essential criteria for a useful screening and measuring method for lead exposure in blood are specificity for lead and sensitivity for physiological responses to lead exposure at concentrations of current concern, i.e. the CDC guideline which defines an elevated blood lead concentration as .gtoreq.10 ug/dL. Additional criteria for a routine screening assay include reliability, ease of performance, low cost, speed, small sample size and the capacity for use in clinical or field settings.